Pathogen Functional Genomics Resource Center, J, Craig Venter Institute, Rockville, MD 20850, USA.
BMC Microbiol. 2011 Jun 24;11:147. doi: 10.1186/1471-2180-11-147.
Shigella dysenteriae serotype 1 (SD1) causes the most severe form of epidemic bacillary dysentery. Quantitative proteome profiling of Shigella dysenteriae serotype 1 (SD1) in vitro (derived from LB cell cultures) and in vivo (derived from gnotobiotic piglets) was performed by 2D-LC-MS/MS and APEX, a label-free computationally modified spectral counting methodology.
Overall, 1761 proteins were quantitated at a 5% FDR (false discovery rate), including 1480 and 1505 from in vitro and in vivo samples, respectively. Identification of 350 cytoplasmic membrane and outer membrane (OM) proteins (38% of in silico predicted SD1 membrane proteome) contributed to the most extensive survey of the Shigella membrane proteome reported so far. Differential protein abundance analysis using statistical tests revealed that SD1 cells switched to an anaerobic energy metabolism under in vivo conditions, resulting in an increase in fermentative, propanoate, butanoate and nitrate metabolism. Abundance increases of transcription activators FNR and Nar supported the notion of a switch from aerobic to anaerobic respiration in the host gut environment. High in vivo abundances of proteins involved in acid resistance (GadB, AdiA) and mixed acid fermentation (PflA/PflB) indicated bacterial survival responses to acid stress, while increased abundance of oxidative stress proteins (YfiD/YfiF/SodB) implied that defense mechanisms against oxygen radicals were mobilized. Proteins involved in peptidoglycan turnover (MurB) were increased, while β-barrel OM proteins (OmpA), OM lipoproteins (NlpD), chaperones involved in OM protein folding pathways (YraP, NlpB) and lipopolysaccharide biosynthesis (Imp) were decreased, suggesting unexpected modulations of the outer membrane/peptidoglycan layers in vivo. Several virulence proteins of the Mxi-Spa type III secretion system and invasion plasmid antigens (Ipa proteins) required for invasion of colonic epithelial cells, and release of bacteria into the host cell cytosol were increased in vivo.
Global proteomic profiling of SD1 comparing in vivo vs. in vitro proteomes revealed differential expression of proteins geared towards survival of the pathogen in the host gut environment, including increased abundance of proteins involved in anaerobic energy respiration, acid resistance and virulence. The immunogenic OspC2, OspC3 and IpgA virulence proteins were detected solely under in vivo conditions, lending credence to their candidacy as potential vaccine targets.
志贺氏痢疾杆菌血清型 1（SD1）引起最严重的爆发性细菌性痢疾。通过二维液相色谱-质谱联用（2D-LC-MS/MS）和 APEX（一种无标记的计算修改的谱计数方法）对志贺氏痢疾杆菌血清型 1（SD1）在体外（来源于 LB 细胞培养物）和体内（来源于无菌小猪）进行了定量蛋白质组学分析。
总体而言，在 FDR（假发现率）为 5%的情况下，共定量了 1761 种蛋白质，其中体外和体内样本分别定量了 1480 种和 1505 种。鉴定了 350 种细胞质膜和外膜（OM）蛋白（占预测的 SD1 膜蛋白组的 38%），这是迄今为止对志贺氏菌膜蛋白组的最广泛调查。使用统计检验进行的差异蛋白丰度分析表明，SD1 细胞在体内条件下切换到厌氧能量代谢，导致发酵、丙酸盐、丁酸盐和硝酸盐代谢增加。转录激活因子 FNR 和 Nar 的丰度增加支持了在宿主肠道环境中从需氧呼吸到厌氧呼吸的转变的观点。与酸应激抗性（GadB、AdiA）和混合酸发酵（PflA/PflB）相关的高体内丰度的蛋白表明细菌对酸应激的存活反应，而增加的氧化应激蛋白（YfiD/YfiF/SodB）表明针对氧自由基的防御机制被动员。涉及肽聚糖周转（MurB）的蛋白增加，而外膜/β-桶 OM 蛋白（OmpA）、OM 脂蛋白（NlpD）、参与 OM 蛋白折叠途径的伴侣（YraP、NlpB）和脂多糖生物合成（Imp）的蛋白减少，表明在体内对外膜/肽聚糖层进行了意想不到的调节。几种 Mxi-Spa 型 III 型分泌系统的毒力蛋白和侵袭质粒抗原（Ipa 蛋白）需要入侵结肠上皮细胞，并将细菌释放到宿主细胞胞质溶胶中，在体内增加。
对 SD1 的体内与体外蛋白质组进行比较的全球蛋白质组学分析显示，与适应宿主肠道环境的生存相关的蛋白质表达存在差异，包括参与厌氧能量呼吸、酸应激抗性和毒力的蛋白丰度增加。免疫原性 OspC2、OspC3 和 IpgA 毒力蛋白仅在体内条件下检测到，这为它们作为潜在疫苗靶标的候选物提供了依据。
